Glutamate, the major excitatory transmitter in the central nervous system is crucial for synaptic transmission and plasticity as well as the pathophysiological process termed "excitotoxicity." Excitotoxicity usually requires activation of a specific glutamate receptor, the N-methyl-D-aspartate (NMDA) receptor. Therapeutic applications of NMDA receptor antagonists in humans though have been unsuccessful due to adverse effects. Better understanding of the modulation of NMDA receptor activity, localization, and turnover may provide novel ways to control excitotoxicity while minimizing deleterious effects of NMDA receptor blockade. NMDA receptor localization and function is controlled by many events including cleavage of the C-terminal of the NR2B subunit by calpain and phosphorylation of NR2B by Src family tyrosine kinases (SFK) (1). Our preliminary data demonstrate that phosphorylation of NR2B by Fyn controls NR2B cleavage by calpain and activation of downstream signaling events including activation of p38 MAK kinase. Activation of Fyn in this paradigm depends on NMDA receptor activation, allowing Y1336 phosphorylation to be part of positive and negative feedback mechanisms controlling neuronal responses. In this proposal, we will dissect the molecular mechanism of the interactions of calpain, Fyn and MAGUK proteins in the control of NMDA receptor properties, and investigate their importance in NMDA receptor physiology and models of excitotoxic mechanisms of human diseases. In aim 1, we will examine the ability of NMDA receptor-activated Fyn to phosphorylate distinct sites on the NMDA receptor and other substrates. This will allow us to understand the mechanism by which NMDA receptors directly or indirectly activate SFK and how such activity is directed to distinct sites on the NMDA receptor. Aim 2 will define the structural determinants and mechanisms that mediate the interactions of calpain (and its subtypes) with NMDA receptors and SFK. In Aim 3, we will assess examine electrophysiological properties of calpain-cleaved NMDA receptors and whether cleavage by calpain alters activation of downstream MAP kinases in order to link further calpain mediated cleavage of NR2B with physiological and pathophysiological events. The final aim will investigate whether the interactions of calpain and SFK alter pathophysiological events in an in vitro model of excitotoxicity. Better understanding of the mechanisms by which calpain modulates NMDA receptors and how MAGUK proteins and SFK alter this process should allow a rational basis for use of agents modulating these events in neurological disorders. PUBLIC HEALTH RELEVANCE: The proposal addresses the mechanisms of degradation of the N-methyl-D-aspartate receptor. Through understanding of this process, the proposal may facilitate therapies for preventing damage in neurologic disorders such as stroke.